Electron transfer across the cell envelope is a critical process in all cellular organisms. Proteins with redox-active cofactors construct insulated circuits that span the cell envelope, connecting cytoplasmic electron pools to electron acceptors in the extracytosolic space. Proteins that use covalently attached flavin mononucleotide moieties (FMN) as redox-active cofactors have been recently reported by several studies to be involved in extracytosolic electron transfer. These proteins are substrates for the FMN transferase ApbE, which post-translationally attaches FMN to its targets through a process termed flavinylation. Yet, there exists a great paucity of information on the prevalence and molecular basis of flavinylated proteins. We aimed to provide a better understanding of the biological and biochemical contexts of ApbE-mediated flavinylation. To this end, we conducted comprehensive studies with three primary goals: 1) evaluate phylogenetic diversity of flavinylated proteins, 2) determine structural and functional properties of flavinylated proteins, and 3) assess relevance of flavinylated proteins to human health.

Our work reveals that ApbE-flavinylated proteins are highly prevalent among prokaryotic organisms, with implicated roles in crucial cellular processes, such as anaerobic respiration or iron assimilation. Comparative genomics and computational structural biology further demonstrate similar versatility of flavinylated proteins in their predicted structures and functions. We identify and experimentally confirm previously unknown proteins as novel ApbE substrates. We also identify flavinylated proteins encoded by pathogenic bacterial species, confirming their FMN-binding activity and mode of electron transfer in vitro. And finally, we provide preliminary assessment of prevalence of flavinylation in publicly available human gut microbiome data. Our work thus expands the breadth of our knowledge on flavinylation-mediated extracytosolic electron transfer, revealing a new, common facet of prokaryotic redox physiology.